The most common bacterial pathogens of plants colonize the apoplast, and from that location outside of the walls of living cells they incite a variety of diseases in most cultivated plants (Alfano et al., xe2x80x9cBacterial Pathogens in Plants: Life Up Against the Wall,xe2x80x9d Plant Cell 8:1683-1698 (1996)). The majority of these are Gram-negative bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia. Most are host specific and will elicit the hypersensitive response (xe2x80x9cHRxe2x80x9d) in nonhosts. The HR is a rapid, programmed death of plant cells in contact with the pathogen. Some of the defense responses associated with the HR are localized at the periphery of plant cells at the site of bacterial contact, but what actually stops bacterial growth is not known (Brown et al., xe2x80x9chrp genes in Xanthomonas campestris pv. vesicatoria Determine Ability to Suppress Papilla Deposition in Pepper Mesophyll Cells,xe2x80x9d MPMI 8:825-836 (1995); Young et al., xe2x80x9cChanges in the Plasma Membrane Distribution of Rice Phospholipase D During Resistant Interactions With Xanthomonas oryzae pv. oryzae,xe2x80x9d Plant Cell 8:1079-1090 (1996); Bestwick et al., xe2x80x9cLocalization of Hydrogen Peroxide Accumulation During the Hypersensitive Reaction of Lettuce Cells to Pseudomonas syringae pv. phaseolicola,xe2x80x9d Plant Cell 9:209-221 (1997)). Pathogenesis in host plants, in contrast, involves prolonged bacterial multiplication, spread to surrounding tissues, and the eventual production of macroscopic symptoms characteristic of the disease. Although these bacteria are diverse in their taxonomy and pathology, they all possess hrp (xe2x80x9chypersensitive response and pathogenicityxe2x80x9d) genes which direct their ability to elicit the HR in nonhosts or to be pathogenic (and parasitic) in hosts (Lindgren, xe2x80x9cThe Role of hrp Genes During Plant-Bacterial Interactions,xe2x80x9d Annu. Rev. Phytopathol. 35:129-152 (1997)). The hrp genes encode a type III protein secretion system that appears to be capable of delivering proteins, known as effector proteins, across the walls and plasma membranes of living plant cells. Such effector proteins are variously known as hypersensitive response elicitors, Avr (Avirulence) proteins, Hop (hypersensitive response and pathogenicity-dependent outer proteins), Vir (virulence) proteins, or Pth (pathogenicity) proteins, depending on the phenotype by which they were discovered (see, e.g., Alfano et al., xe2x80x9cThe Type III (Hrp) Secretion Pathway of Plant Pathogenic Bacteria: Trafficking Harpins, Avr Proteins, and Death,xe2x80x9d J. Bacteriol. 179:5655-5662 (1997), which is hereby incorporated by reference). The Avr proteins are so named because they can betray the parasite to the R gene-encoded surveillance system of plants, thereby triggering the HR (Vivian et al., xe2x80x9cAvirulence Genes in Plant-Pathogenic Bacteria: Signals or Weapons?,xe2x80x9d Microbiology 143:693-704 (1997); Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annul. Rev. Phytopathol. 34:153-179 (1996)). But Avr-like proteins also appear to be key to parasitism in compatible host plants, where the parasite proteins are undetected and the HR is not triggered. Thus, bacterial avirulence and pathogenicity are interrelated phenomena and explorations of HR elicitation are furthering our understanding of parasitic mechanisms.
A current model for plant-bacterium interaction and co-evolution based on Hrp delivery of Avr proteins into plant cells proposes that (i) Avr-like proteins are the primary effectors of parasitism, (ii) conserved Hrp systems are capable of delivering many, diverse Avr-like proteins into plant cells, and (iii) genetic changes in host populations that reduce the parasitic benefit of an effector protein or allow its recognition by the R-gene surveillance system will lead to a proliferation of complex arsenals of avr-like genes in co-evolving bacteria (Alfano et al., xe2x80x9cBacterial Pathogens in Plants: Life Up Against the Wall,xe2x80x9d Plant Cell, 8:1683-1698 (1996)). There are still many gaps in this model. For example, the physical transfer of Avr proteins into plant cells has never been observed, the virulence functions of Avr proteins are unknown, and it is likely that previous searches for Avr genes in various bacteria have yielded incomplete inventories of the genes in various bacteria and, thus, incomplete inventories of the genes encoding effector proteins.
Until recently, Avr proteins had not been reported outside of the cytoplasm of living Pseudomonas syringae and Xanthomonas spp. cells (Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annul. Rev. Phytopathol, 34:153-179 (1996); Puri et al., xe2x80x9cExpression of avrPphB, an Avirulence Gene from Pseudomonas Syringae pv. phaseolicola, and the Delivery of Signals Causing the Hypersensitive Reaction in Bean,xe2x80x9d MPMI 10:247-256 (1997)), but it now appears that the Hrp systems of Erwinia spp. can secrete Avr proteins in culture. A homolog of the Pseudomonas syringae pv. tomato avrE gene has been found in Erwinia amylovora and designated dspA in strain CFBP1430 and dspE in strain Ea321 (Gaudriault et al., xe2x80x9cDspA, an Essential Pathogenicity Factor of Erwinia amylovora Showing Homology with AvrE of Pseudomonas syringae, is Secreted via the Hrp Secretion Pathway in a DspB-dependent Way,xe2x80x9d Mol. Microbiol., 26:1057-1069 (1997); Bogdanove et al., xe2x80x9cHomology and Functional Similarity of a hrp-linked Pathogenicity Operon, dspEF, of Erwinia amylovora and the avrE locus of Pseudomonas syringae Pathovar Tomato,xe2x80x9d Proc. Natl. Acad. Sci. USA, 95:1325-1330 (1998)). dsp genes are required for the pathogenicity of Erwinia amylovora, but not for HR elicitation. A protein of the expected size of DspA is secreted in a Hrp- and DspB-dependent manner by CFBP1430 (DspB is a potential chaperone) (Gaudriault et al., xe2x80x9cDspA, an Essential Pathogenicity Factor of Erwinia amylovora Showing Homology with AvrE of Pseudomonas syringae, is Secreted via the Hrp Secretion Pathway in a DspB-dependent Way,xe2x80x9d Mol. Microbiol., 26:1057-1069 (1997)). Specific antibodies were used to demonstrate unambiguously that DspE is efficiently secreted in a Hrp-dependent manner by strain Ea321 (Bogdanove et al., xe2x80x9cErwinia amylovora Secretes DspE, a Pathogenicity Factor and Functional AvrE Homolog, Through the Hrp (Type III Secretion) Pathway,xe2x80x9d J. Bacteriol., 180(8):2244-2247 (1998)).
Furthermore, the Erwinia chrysanthemi Hrp system enables E. coli to secrete effector proteins of P. syringae and Yersinia spp. (Ham, et al., xe2x80x9cA Cloned Erwinia chrysanthemi Hrp (Type III Protein Secretion) System Functions in Escherichia coli to Deliver Pseudomonas syringae Avr Signals to Plant Cells and to Secrete Avr Proteins in Culture,xe2x80x9d Proc. Natl. Acad. Sci. USA 95:10206-10211 (1998); Anderson et al., xe2x80x9cReciprocal Secretion of Proteins by the Bacterial Type III Machines of Plant and Animal Pathogens Suggests Universal Recognition of mRNA Targeting Signals,xe2x80x9d Proc. Natl. Acad. Sci. USA 96:12839-12843 (1999); Mudgett and Staskawicz, xe2x80x9cCharacterization of the Pseudomonas syringae pv. tomato AvrRpt2 Protein: Demonstration of Secretion and Processing During Bacterial Pathogenesis,xe2x80x9d Mol. Microbiol. 32:927-941 (1999)). Also, conditions have now been defined that permit detection of Hrp-dependent secretion of effector proteins by P. syringae and X. campestris. Rossier et al., xe2x80x9cThe Xanthomonas Hrp Type III System Secretes Proteins from Plant and Mammalian Bacterial Pathogens,xe2x80x9d Proc. Natl. Acad. Sci. USA 96:9368-9373 (1999); van Dijk et al., xe2x80x9cThe Avr (Effector) Proteins HrmA (HopPsyA) and AvrPto are Secreted in Culture from Pseudomonas syringae Pathovars via the Hrp (Type III) Protein Secretion System in a Temperature and pH-Sensitive Manner,xe2x80x9d J. Bacteriol. 181:4790-4797 (1999)).
The biochemical activities or parasite-promoting functions of effector proteins remain unclear, although several of those known make measurable contributions to virulence (Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annul. Rev. Phytopathol, 34:153-179 (1996)). Members of the AvrBs3 family in Xanthomonas spp. are targeted to the plant nucleus (Van den Ackerveken et al., xe2x80x9cBacterial Avirulence Proteins as Triggers of Plant Defense Resistance,xe2x80x9d Trends Microbiol, (1997); Gabriel, xe2x80x9cTargeting of Protein Signals from Xanthomonas to the Plant Nucleus,xe2x80x9d Trends Plant Sci., 2:204-206 (1997)), and some of these have been shown recently to redundantly encode watersoaking functions associated with circulence (Yang et al., xe2x80x9cWatersoaking Function(s) of XcmH1005 are Redundantly Encoded by Members of the Xanthomonas avr/pth Gene Family,xe2x80x9d MPMI, 9:105-113 (1996)). AvrD (Pseudomonas syringae pv. tomato) directs the synthesis of syringolide elicitors of the HR (Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annul. Rev. Phytopathol, 34:153-179 (1996)); AvrBs2 (Xanthomonas campestris pv. vesicatoria) shows similarity to A. tumefaciens agrocinopine synthase (Swords et al., xe2x80x9cSpontaneous and Induced Mutations in a Single Open Reading Frame Alters Both Virulence and Avirulence in Xanthomonas campestris pv. vesicatoria avrBs2,xe2x80x9d J. Bacteriol., 4661-4669 (1996)); and AvrRxv (Xanthomonas campestris pv. vesicatoria) is a homolog of AvrA (Salmonella typhimurium) and YopJ (Yersinia spp.), proteins which travel the type III pathway in animal pathogens and trigger apoptosis in macrophages (Hardt et al., xe2x80x9cA Secreted Salmonella Protein With Homology to an Avirulence Determinant of Plant Pathogenic Bacteria,xe2x80x9d Proc. Natl. Acad. Sci. USA, 94:9887-9892 (1997); Monack et al., Yersinia Signals Macrophages to Undergo Apoptosis and YopJ is Necessary for this Cell Death,xe2x80x9d Proc. Natl. Acad. Sci. USA, 94:10385-10390 (1997)). This last observation has led to the suggestion that avr-R gene interactions may occur also in animal pathogenesis (Galan, xe2x80x9cxe2x80x98Avirulence Genexe2x80x99 in Animal Pathogens?,xe2x80x9d Trends Microbiol., 6:3-6 (1998)).
The primary sequences of the Pseudomonas syringae Avr proteins reveal little about their potential function, but interestingly, when heterologously expressed in plants, three of them have produced necrosis in test plants lacking the cognate R gene (Gopalan et al., xe2x80x9cExpression of the Pseudomonas syringae Avirulence Protein AvrB in Plant Cells Alleviates its Dependence on the Hypersensitive Response and Pathogenicity (Hrp) Secretion System in Eliciting Genotype-specific Hypersensitive Cell Death,xe2x80x9d Plant Cell, 8:1095-1105 (1996); Stevens et al., xe2x80x9cSequence Variations in Alleles of the Avirulence Gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola Lead to Loss of Recognition of the AvrPphE Protein Within Bean Cells and Gain in Cultivar Specific Virulence,xe2x80x9d Mol. Microbiol., 29(1):165-77 (1998); McNellis et al., xe2x80x9cGlucocorticoid-inducible Expression of a Bacterial Avirulence Gene in Transgenic Arabidopsis Induces Hypersensitive Cell Death,xe2x80x9d Plant J., 14(2):247-57 (1998)). A key question is whether this results from interaction of abnormally high levels of the bacterial protein with plant virulence targets or with cross-reacting R-gene products. Further evidence suggesting that some avr genes in Pseudomonas syringae are beneficial to the bacteria in host plants is found in recent studies of avrD and avrPphE. Highly conserved, nonfunctional alleles of these genes have been retained in pathogens whose hosts would recognize the functional Avr product (Stevens et al., xe2x80x9cSequence Variations in Alleles of the Avirulence Gene avrPphE.R2 from Pseudomonas syringae pv. phaseolicola Lead to Loss of Recognition of the AvrPphE Protein Within Bean Cells and Gain in Cultivar Specific Virulence,xe2x80x9d Mol. Microbiol., 29(1):165-77 (1998); Keith et al., xe2x80x9cComparison of avrD Alleles from Pseudomonas syringae pv. glycinea,xe2x80x9d MPMI, 10:416-422 (1997)).
Avr-like genes may function heterologously to support pathogenesis as well as HR elicitation. The pathogenicity of an Erwinia amylovora dspE mutant can be restored (at least partially) by a plasmid carrying the Pseudomonas syringae avrE locus, suggesting that DspE and AvrE have similar functions (Bogdanove et al., xe2x80x9cHomology and Functional Similarity of a hrp-linked Pathogenicity Operon, dspEF, of Erwinia amylovora and the avrE locus of Pseudomonas syringae Pathovar Tomato,xe2x80x9d Proc. Natl. Acad. Sci. USA, 95:1325-1330 (1998)). That dspE is essential for Erwinia amylovora pathogenicity, whereas avrE contributes only quantitatively to the virulence of Pseudomonas syringae pv tomato (Lorang et al., xe2x80x9cavrA and avrE in Pseudomonas syringae pv. Tomato PT23 Play a Role in Virulence on Tomato Plants,xe2x80x9d MPMI, 7:508-515 (1994)), suggests that there is less redundancy in the Erwinia amylovora virulence system. This would be consistent with a more recent acquisition of the Hrp system by Erwinia amylovora and/or a slower coevolution with its perennial hosts (Bogdanove et al., xe2x80x9cHomology and Functional Similarity of a hrp-linked Pathogenicity Operon, dspEF, of Erwinia amylovora and the avrE locus of Pseudomonas syringae Pathovar Tomato,xe2x80x9d Proc. Natl. Acad. Sci. USA, 95:1325-1330 (1998)). The heterologous function of Pseudomonas syringae avr genes in Erwinia amylovora and Erwinia chrysanthemi suggests that Hrp+ bacteria in the field may be able to xe2x80x98samplexe2x80x99 a buffet of avr-like genes from diverse sources in their coevolution with changing plant populations. Many avr genes have been known to be potentially mobile, because of their presence on plasmids (Vivian et al., xe2x80x9cAvirulence Genes in Plant-Pathogenic Bacteria: Signals or Weapons?,xe2x80x9d Microbiology 143:693-704 (1997); Leach et al., xe2x80x9cBacterial Avirulence Genes,xe2x80x9d Annu. Rev. Phytopathol, 34:153-179 (1996)). Recent observations with Pseudomonas syringae highlight the apparent mobility of avr genes. Several Pseudomonas syringae avr genes are liked with transposable elements or phage sequences (Hanekamp et al., xe2x80x9cAvirulence Gene D of Pseudomonas syringae pv. Tomato May Have Undergone Horizontal Gene Transfer,xe2x80x9d FEBS Lett., 415:40-44 (1997)), and the hrp clusters in different strains of Pseudomonas syringae, although conserved in themselves, are bordered by a hypervariable region enriched in avr genes and mobile DNA elements. Alfano et al., xe2x80x9cThe Pseudomonas syringae Hrp Pathogenicity Island has a Tripartite Mosaic Structure Composed of a Cluster of Type III Secretion Genes Bounded by Exchangeable Effector and Conserved Effector Loci that Contribute to Parasitic Fitness and Pathogenicity in Plants,xe2x80x9d Proc. Natl. Acad. Sci. USA 97:4856-4861 (2000).
Two classes of extracellular Hrp proteins have now been defined-harpins and pilins. Harpins are glycine-rich proteins that lack cysteine, are secreted in culture when the Hrp system is expressed, and possess heat-stable HR elicitor activity when infiltrated into the leaves of tobacco and several other plants (Alfano et al., xe2x80x9cBacterial Pathogens in Plants: Life Up Against the Wall,xe2x80x9d Plant Cell, 8:1683-1698 (1996)). Mutation of the prototypical hrpN harpin gene in Erwinia amylovora Ea321 strongly diminishes HR and pathogenicity phenotypes (Kim et al., xe2x80x9cHrpW of Erwinia amylovora, a New Harpin That is a Member of a Proposed Class of Pectate Lyases,xe2x80x9d J. Bacteriol. 180(19):5203-5210 (1998)), but mutation of the hrpZ harpin gene in different Pseudomonas syringae strains has little or no effect on Hrp phenotypes (Alfano et al., xe2x80x9cAnalysis of the Role of the Pseudomonas syringae pv. syringae HrpZ Harpin in Elicitation of the Hypersensitive Response in Tobacco Using Functionally Nonpolar Deletion Mutations, Truncated HrpZ Fragments, and hrmA Mutations,xe2x80x9d Mol. Microbiol. 19:715-728 (1996); Charkowski et al.,. xe2x80x9cThe Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate,xe2x80x9d J. Bacteriol. 180 (19):5211-5217 (1998)). The natural function of harpins or the basis for their ability to elicit an apparent programmed cell death when artificially introduced into the apoplast of plants is unknown. However, two lines of evidence point to a site of action in the plant cell wall. First, purified Pseudomonas syringae harpin binds to cell walls and has biological activity only with walled cells (Hoyos et al., xe2x80x9cThe Interaction of HarpinPss With Plant Cell Walls,xe2x80x9d MPMI 9:608-616 (1996)). Second, HrpW, a second harpin discovered in both Erwinia amylovora and Pseudomonas syringae, has an N-terminal half that is harpin-like but a C-terminal half that is homologous to a newly-defined class of pectate lyases found in fungal and bacterial pathogens (Kim et al., xe2x80x9cHrpW of Erwinia amylovora, a New Harpin That is a Member of a Proposed Class of Pectate Lyases,xe2x80x9d J. Bacteriol. 180(19):5203-5210 (1998); Charkowski et al., xe2x80x9cThe Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate,xe2x80x9d J. Bacteriol. 180 (19):5211-5217 (1998)). Elicitor activity resides in the harpin domain, and the pectate lyase domain, although lacking enzymatic activity, binds specifically to pectate (Charkowski, A. et al., xe2x80x9cThe Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate,xe2x80x9d J. Bacteriol. 180 (19):5211-5217 (1998)). The second class of extracellular Hrp proteins are represented by the Pseudomonas syringae HrpA pilin, which is a subunit of a Hrp-pilus that is 6-8 nm in diameter and is formed on bacteria in a Hrp-dependent manner (Roine et al., xe2x80x9cHrp Pilus: An hrp-dependent Bacterial Surface Appendage Produced by Pseudomonas syringae pv. tomato DC3000, xe2x80x9d Proc. Natl. Acad. Sci. USA 94:3459-3464 (1997)). The Hrp pilus is required for pathogenicity and elicitation of the HR, and a similar structure is important for T-DNA transfer in Agrobacterium tumefaciens (Fullner et al., xe2x80x9cPilus Assembly by Agrobacterium T-DNA Transfer Genes,xe2x80x9d Science, 237:1107-1109 (1996)). Whether these structures promote the transfer of bacterial macromolecules into plant cells by serving as conduits, guides, or attachment factors is not known.
Type III secretion systems are present in both animal and plant pathogenic bacteria, which indicates that they are capable of operating not only across bacterial genera but also across host kingdoms (Galan et al., xe2x80x9cType III Secretion Machines: Bacterial Devices for Protein Delivery into Host Cells,xe2x80x9d Science 284:1322-1328 (1999)). At present, the metabolic changes caused by effector proteins secreted by the type III protein secretion system of plant pathogenic bacteria are unknown. However, perturbations in pathways involved in innate immunity, programmed cell death, and the cell cycle are unlikely. Supporting this expectation is the finding that effectors of Salmonella, Shigella, and Yersinia spp. have activities such as altering F-actin stability, activation of caspase-1, tyrosine phosphatase activity, and inhibition of mitogen-activated protein kinases (Galxc3xa1n et al., xe2x80x9cType III Secretion Machines: Ingenious Bacterial Devices for Protein Delivery into Host Cells,xe2x80x9d Science 284:1322-1328 (1999); Orth et al., xe2x80x9cInhibition of the Mitogen-Activated Protein Kinase Superfamily by a Yersinia Effector,xe2x80x9d Science 285:1920-1923 (1999)). Many of the metabolic targets are likely to be universal among eucaryotes and, therefore, these phytopathogen effector proteins are likely to provide tools for altering the metabolism of yeast, nematodes, insects, and higher animals for various biotechnological purposes.
A limiting factor in the potential biotechnological use of these phytopathogen effector proteins is that the metabolic targets of the effector proteins are inside host cells and, therefore, the effector proteins must be either produced inside the target cells or delivered into them by some means. One such means is gene therapy techniques, however, this technology is relatively difficult to apply.
Thus, it would be beneficial to obtain a recombinant construct and delivery system which overcomes these and other deficiencies in the art.
One aspect of the present invention relates to a method for delivering effector proteins into a target cell. This method involves introducing into the target cell an effector protein fused to a protein transduction domain of a human immunodeficiency virus TAT protein or derivatives or functional analogs thereof.
Another aspect of the present invention relates to a DNA construct including a first DNA molecule encoding an effector protein and a second DNA molecule operatively associated with the first DNA molecule and encoding a protein transduction domain of a human immunodeficiency virus TAT protein or derivatives or functional analogs thereof.
The method of the present invention allows efficient delivery of effector proteins into cells, in particular, mammalian cells. This method also allows for delivery of effector proteins for use in pharmaceutical, insecticide, fungicide, herbicide, and other applications. In particular, the present invention will allow the delivery of effector proteins into patients in the form of protein therapy. Therapy with biologically active full-length proteins will allow access to the built-in evolutionary specificity of these proteins for their targets, thereby potentially avoiding the nonspecific effects sometimes seen with small-molecule therapies. Moreover, when used in conjunction with tissue-specific viral vectors, use of the present invention allows the targeted delivery of effector proteins to particular cells with the added benefit of secondary redistribution of the effector protein subsequent to the initial targeting. A precedent for this approach can be found in an experiment wherein the VP22 protein transduction domain was fused to the p53 tumor suppressor protein (Phelan et al., xe2x80x9cIntercellular Delivery of Functional p53 by the Herpesvirus Protein VP22,xe2x80x9d Nat. Biotechnol. 16:440-443 (1998), which is hereby incorporated by reference).